THE VACCINIA VIRUS-STIMULATED PATHWAYS MEK/ERK AND JNK LEADING TO c-JUN ACTIVATION IS CRITICAL FOR VIRAL BIOLOGY.
Pereira, A.C.T.C.; Leite, F.G.G.; Brasil, B.S.A.F.; Andrade, A.A.A.; Kroon, E.G.; Ferreira, P.C.P.; Bonjardim, C.A.
Laboratório de Vírus, Departamento de Microbiologia, Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais.
Vaccinia virus (VV) is a large DNA virus that multiplicates in the cytoplasm of host infected cells. We have previously shown that the mitogen-activated protein kinase (MAPK) pathway MEK/ERK1/2 is required for VV multiplication (Andrade et. Al., 2004). This observation was then extended to the participation of another pathway involving the stress-activated protein kinases (SAPKs) JNK1/2–c-JUN as necessary for maximal VV multiplication (Pereira et al, manuscript in preparation). Here we show that VV infection coordinately regulates ERK1/2 and JNK1/2 pathways and leads to c-JUN activation during the whole virus infective cycle. Experiments were carried out after depriving the A31 cells with 1% of fetal bovine serum for 12h and then virus infected at multiplicity of infection 10 in the absence or presence of MEK or JNK inhibitors (PD98059 or UO126) or (SP600125), respectively. Cell lysates were then prepared and immunoblotted with anti-phospho: ERK1/2, JNK1/2 and c-JUN antibodies. Our data show that c-JUN is phosphorylated at early and intermediate times of the infection (3-24 hpi) mainly by ERK1/2, whereas JNK phosphorylates it at late times (30-48 hpi). Because c-JUN once activated may act as a transcriptional regulator, we next investigated whether it interacted with the cis-acting regulatory sequences AP-1 or CRE found at the regulatory region of AP-1- or CRE-dependent genes To that end, cells were virus-infected for different times and cellular protein extracts were prepared and the DNA-protein complexes formed were analyzed by EMSA. An increase in AP-1-c-JUN protein complex formed was observed upon VV infection at 5 and 24 hpi, which was drastically affected by pre-incubation with UO126. In agreement with the differential requirement of MEK/ERK and JNK to activate c-JUN, CRE-c-JUN complex formation at 30 hpi was dependent on JNK, since pre-incubation with SP600125 abolished DNA-protein interaction. These pathways appear to be of biological relevance because viral yield was drastically decreased when the multiplication was carried out on a cell line expressing c-JUN dominant-negative mutation.
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